Vol. 41, No. 2, 2004
Issue release date: March–April 2004
J Vasc Res 2004;41:202–207
Research Paper
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Exogenous BH4/Bcl-2 Peptide Reverts Coronary Endothelial Cell Apoptosis Induced by Oxidative Stress

Cantara S.a · Donnini S.a · Giachetti A.a,b · Thorpe P.E.c · Ziche M.a
aPharmacology Section, Department of Molecular Biology, University of Siena, Siena, and bLifetech s.r.l., Florence, Italy; cDepartment of Pharmacology and Simmons Cancer Center, University of Texas Southwestern Medical Center, Dallas, Tex., USA
email Corresponding Author


 goto top of outline Key Words

  • BH4/BcL-2
  • Apoptosis
  • Oxidative stress
  • Endothelium

 goto top of outline Abstract

Background: Vascular endothelium undergoes apoptosis when exposed to reactive oxygen species (ROS), including hydrogen peroxide and superoxide radicals. ROS are believed to be the cause of damage to small vessels during ischemia-reperfusion injury and of arterial damage during atherosclerosis. Hydrogen peroxide-induced apoptosis is mediated through the inhibition of Bcl-xl activity and caspase-3 and caspase-9 activation. The BH4 domain of the Bcl-2 family members is responsible for their antiapoptotic activity. The BH4 domains of Bcl-2 and Bcl-xl inhibit cytochrome c release and the loss of mitochondrial membrane potential. Methods and Results: The purpose of this project was to study the antiapoptotic effect of cell-permeant derivative of Bcl-2 (BH4 peptide) on endothelial cells exposed to stress conditions. BH4 peptide was conjugated to the cell-permeable peptide TAT and was applied to endothelial cells under conditions of serum starvation and hydrogen peroxide treatment. TAT-BH4 reduced caspase-3 activity and prevented apoptotic cell death. Conclusion: Our results indicate that TAT-BH4 peptide can protect endothelial cells from ROS-induced apoptosis.

Copyright © 2004 S. Karger AG, Basel

goto top of outline Introduction

Vascular endothelium undergoes apoptosis when exposed to reactive oxygen species (ROS), including hydrogen peroxide and superoxide radicals [1, 2, 3]. Apoptosis is characterized by a series of morphological changes, including chromatin condensation, membrane blebbing, cell shrinkage, cell fragmentation and death [4]. The apoptotic process is executed by cysteine proteases (caspases) and regulated by proapoptotic and antiapoptotic members of the Bcl-2 protein family [5]. The proteins of this family are characterized by four different homology domains called Bcl-2 homology (BH) domains: BH1, BH2, BH3 and BH4 [5, 6], of which only BH4 has antiapoptotic properties [7, 8]. BH4 is one of the four different homology domains of the Bcl-2 protein, which has been shown to block Ras-mediated apoptotic activity as well as cytochrome c release and the mitochondrial membrane potential loss [7, 8, 9].

Here we report preliminary studies in coronary endothelial cells (CVEC) in which apoptosis, induced by exposure to graded concentrations of hydrogen peroxide in a low serum concentration, was followed by investigating the cell morphology through specific staining and by studying their proliferation in terms of survival. The biochemical mechanisms underlying endothelial cell apoptosis were examined by determining the activity of caspase-3, the terminal enzyme of the proteolytic caspase cascade responsible for cell death. In addition, we evaluated the ability of BH4, administrated to cells as the conjugated form with the cell-permeable peptide TAT (TAT BH4), to counteract the endothelial cell apoptotic process induced by hydrogen peroxide.


goto top of outline Materials and Methods

goto top of outline Cell Culture

CVEC were cultured in 10% bovine calf serum (BCS; Hyclone) as previously described [10]. Human umbilical vein endothelial cells (HUVEC) were cultured as described [11]. Porcine aortic endothelial cells (PAEC) were cultured as reported [12]. Cells between passage 15 and 20 for CVEC and PAEC, and between passage 3 and 5 for HUVEC were used in the experiments.

goto top of outline Caspase-3 Activity

The caspase-3 activity was measured using EnzCheck® Caspase-3 Assay kit No. 2 (Molecular Probes Europe BV, Leiden, The Netherlands). 4 × 105 cells were plated in 10% serum, starved (0.1% serum) overnight after adherence, and then stimulated with test substances (50 μM H2O2, 50 nM TAT-BH4, 50 nM scrambled peptide and 1% BCS) for 4 h. TAT-BH4 and H2O2 were added at the same time. Cells were harvested with trypsin-EDTA, lysed and centrifuged. To each 50 μl of the lysis supernatants, transferred to individual 96 microplate wells, were added 50 μl of a 2× solution containing 5 mM Z-DEVD-R110 substrate. The microplate, protected from light, was incubated at room temperature for 30 min. The fluorescence was measured by using SpectraFluor (Tecan; excitation/emission 485/535) every 30 min within 3 h.

goto top of outline Fluorescence Microscopy

To detect apoptosis, cells were stained either with 4′,6′-diaminidino phenylidole (DAPI) or with acridine orange/ethidium bromide (AO/EB); 5,000 cells/well were seeded in a 48-multiwell plate and treated with 50 μM H2O2 and/or 50 nM TAT-BH4 for 4 h at 37°C. For the DAPI procedure, cells were incubated with DAPI [1 μg/ml phosphate-buffered saline (PBS)] for 20 min after fixation in 10% paraformaldehyde for 10 min. Cells were examined by fluorescence microscopy at 100× magnification using the appropriate DAPI filter. For the AO/EB procedure, cells were harvested with trypsin-EDTA, centrifuged once in DMEM and later washed with PBS. The pellet was resuspended in 100 μl of PBS. Twenty microliters of cell suspension were combined with AO/EB working solution prepared by combining 10 μg/ml of both AO and EB in 0.9% saline. The slides, prepared by placing 10 μl of the cell/dye mixtures, were examined with a 40× dry objective, using the fluorescence microscope (Eclipse TE300; Nikon) and the appropriate fluorescein filter. Every slide was randomly photographed 4 times and the photographs (camera Colpex; Nikon) were reported by using the Adobe Photoshop 5.0 program.

goto top of outline Cell Growth

1.5 × 103 cells resuspended in 10% serum were seeded in 96-multiwell plates. After adherence, cells were serum starved (0.1% serum) for 24 h in order to synchronize cells and to induce a low level of apoptosis, and then stimulated with test substances (25, 50 and 200 μM H2O2, 50 nM TAT-BH4 peptide and 50 nM scrambled peptide). After 48 h, cells were fixed in 100% methanol and stained with Diff-Quik (Mertz-Dade). The total cell number/well was counted in a blinded manner at 10× magnification.

goto top of outline Reagents

H2O2 and cell culture reagents were from Sigma Chemical Co., St. Louis, Mo., USA. TAT-BH4 was obtained from Oncogene (TAT49–57 β-ala-BH44–23). Scrambled peptide TAT49–57-(SKQVLYNSGRVELKYDFSLS) was synthesized in collaboration with Prof. M. Taddei (Dipartimento Farmaco Chimico Tecnologico, University of Siena, Italy).

goto top of outline Statistical Analysis

Results are expressed as means ± SEM for (n) experiments. Statistical analyses were performed using Student’s t test.


goto top of outline Results

goto top of outline Hydrogen Peroxide Impairs Endothelial Cell Viability

Cultured endothelial cells were sensitive to suboptimal serum concentration (0.1% serum) showing a marked decrease of cell count during the 48-hour incubation (approximately 50%) relative to optimal conditions (10% serum; CVEC: 4,300 ± 135 cells in 10% serum vs. 2,130 ± 150 cells in 0.1% serum; HUVEC: 3,980 ± 97 cells in 10% serum vs. 1,978 ± 154 cells in 0.1% serum; PAEC: 4,768 ± 211 cells in 10% serum vs. 2,345 ± 190 cells in 0.1% serum).

Effects comparable to those shown in figure 1 for CVEC (the only ones reported for clarity’s sake) were produced by exposure either to low serum or H2O2 in other endothelial cell lines (HUVEC and PAEC). Exposure of endothelial cells, grown in suboptimal conditions (0.1% serum), to graded concentrations of H2O2 (25, 50 and 200 μM) produced further significant decreases of cell count, indicating the sensitivity of these cells to ROS over and above that of serum deprivation. 50 μM H2O2 was selected as most suitable for further experiments since it consistently inhibited growth by 60% across cell lines (table 1).


Fig. 1. Postcapillary (CVEC) endothelial cells after exposure to increasing concentrations of H2O2 (25, 50 and 200 μM) for 48 h. Data are reported as percentage of basal response ± SEM (n = 4 experiments run in triplicate). + p < 0.0001 vs. 0.1% BCS; * p < 0.001 vs. basal condition (0.1% serum) by Student’s t test.


Table 1. Endothelial cell number inresponse to 50 μM H2O2 alone or incombination with 50 nM TAT-BH4during 48 h culture

Morphological changes of CVEC cultured in 0.1% serum and exposed to 50 μM H2O2 are depicted in figure 2b–e. Changes typical of cells entering apoptosis, i.e. chromatin condensation, membrane blebbing, DNA fragmentation, and cell shrinkage, were readily seen after incubation with H2O2.


Fig. 2. Blockageof apoptosis by TAT-BH4. a Caspase-3 activity in CVEC induced by treatment with 50 μM H2O2. Data are reported as relative fluorescence/mg protein ± SEM (n = 3 run in triplicate). * p < 0.05, ** p < 0.01 vs. basal condition (0.1% serum). bg Changes typical of cells entering apoptosis after AO/EB orDAPI staining, respectively. b, c Control situation (0.1% serum). d, e Endothelial cells after treatment with 50 μM H2O2. f, g Prevention of apoptosis after administration of 50 nM TAT-BH4. Arrows indicate cells in apoptosis evidenced by the two dyes. Asterisks indicate TAT-BH4-treated cells in which apoptosis was not executed, as suggested by intermediate staining.

goto top of outline Caspase-3 Activity Induced by H2O2 and Its Reversal by TAT-BH4

Given the large reduction of cell number and the distinct morphological changes indicative of apoptosis produced by serum deprivation and H2O2 treatment, we deemed it of interest to measure the intracellular level of caspase-3 activity recognized as a reliable biochemical correlate of apoptotic events. The results displayed in figure 2a (only CVEC shown) demonstrate a large increase (nearly 3-fold) of enzyme activity in serum-deprived cells compared to control cells (1% serum). A still larger increase was observed in cells incubated with H2O2 (p < 0.05). The addition of the antiapoptotic peptide BH4, in its permeable form TAT-BH4 (50 nM), completely reversed (p < 0.01) the H2O2 effect on caspase activity (p < 0.01), and reduced the increase produced by low serum. The application of the peptide in its scrambled sequence coupled with TAT (50 nM) was devoid of efficacy on enzyme activity.

Figure 2f and g provide morphological evidence for the antiapoptotic effects exerted by TAT-BH4 by showing restoration of cell profiles similar to the initial ones (fig. 2b, c).

goto top of outline TAT-BH4 Peptide Rescues Cells from Apoptosis

In the light of the reversal exerted by TAT-BH4 on the increased caspase-3 activity promoted by H2O2, it was of interest to investigate whether the peptide could similarly preserve the endothelial cell number after H2O2 treatment. As shown in table 1, application of TAT-BH4 (50 nM) completely rescued endothelial cells (all types) from the evenly severe damage (more than 50% reduction in cell number) produced by H2O2 (50 μM). Cell count after 50 nM TAT-BH4 was at levels slightly above control (CVEC: 2,315 ± 170 cells in H2O2 + TAT-BH4 vs. 2,130 ± 150 cells in 0.1% serum; HUVEC: 2,273 ± 120 cells in H2O2 + TAT-BH4 vs. 1,978 ± 154 cells in 0.1% serum; PAEC: 2,784 ± 130 cells in H2O2 + TAT-BH4 vs. 2,345 ± 190 cells in 0.1% serum). The scrambled peptide (50 nM) exerted no rescuing effect indicating the specificity of the TAT-BH4 effect. Additional experiments on CVEC have shown that the rescuing effect of the TAT-BH4 is concentration dependent, the concentration of 50 nM being submaximal (0.1 nM TAT-BH4 + 50 μM H2O2: 930 ± 167 cells; 5 nM TAT-BH4 + 50 μM H2O2: 1,676 ± 103 cells; 100 nM TAT-BH4 + 50 μM H2O2: 2,387 ± 97 vs.50 μM H2O2: 852 ± 130 cells).


goto top of outline Discussion

Ischemia causes a well-characterized dysfunction of the coronary endothelium through the production of ROS. The therapy of endothelial cell dysfunction offers a wide range of possibilities for interventions. Among these, molecules targeting apoptosis are an area of intense investigation, since programmed cell death has been recognized as an important determinant of vascular endothelial injury [2, 3, 4, 6, 13]. This study contributes to this area of research, as it shows that exogenous administration of BH4, a peptide domain of the antiapoptotic protein Bcl-2, is capable of reversing apoptosis of endothelial cells.

Apoptosis promoted by H2O2 in CVEC was clearly evidenced by the morphological studies showing marked chromatin condensation, cell shrinkage and cell loss detectable by fluorescent stainings specific for apoptosis. In addition, the large elevation of caspase-3 activity, the known effector of apoptotic message, confirmed the occurrence of programmed cell death in endothelial cells.

Exogenous application of BH4, in its cell-permeable form TAT-BH4, to cells protected endothelial cells from undergoing apoptosis in response to serum starvation and hydrogen peroxide treatment. Thus, upon exposure to TAT-BH4, the elevated caspase-3 enzyme activity returned to the control level, cells resumed their morphological profile and their integrity. More importantly, TAT-BH4 prevented endothelial cells from entering the H2O2-induced suicide program, totally restoring their viability, their depleted population and their inherent capacity to proliferate. The effect of TAT-BH4 on promoting cell protection and growth, observed at nanomolar concentrations (50 nM), appeared to be specific since a scrambled sequence of the peptide was devoid of protective activity. In addition, our results indicate that the concentration of TAT-BH4 used in these experiments might be submaximal.

The mechanism of the antiapoptotic effect exerted by TAT-BH4, delineated in the seminal work of Shimizu et al. [7], involves the inhibition of a mitochondrial voltage-dependent ion channel resulting in defective mitochondrial permeability and in the massive release of the apoptogenic cytochrome c. The work of Shimizu et al. also evidenced the antiapoptotic effect of exogenous TAT-BH4 in a tumor cell line (HELA) grown in suspension. Here we show for the first time that exposure to exogenous TAT-BH4 prevents cell loss and maintains the integrity of endothelial cells of diverse lineage. Although the antiapoptotic effect exerted by TAT-BH4 appears to be similar in HELA and endothelial cells, the two cell populations differ substantially in their propensity to initiate a cell death program. In fact, while in HELA TAT-BH4 appears to merely sustain an inherently strong antiapoptotic program, in the endothelial cells, known for their sensitivity to external environment, the peptide appears to provide a survival advantage capable of overriding the cytotoxic insult. Since the antiapoptotic effect of Bcl-2/Bcl-xl family members have been clearly shown to be independent from scavenging activity [14, 15], it is likely that the rescuing effects of TAT-BH4 are not linked to antioxidant activity.

The knowledge that a relatively small peptide (20 amino acids) can prevent the pervasive damaging effect of ROS might have implications for the design of novel therapies for cardiovascular pathologies which recognize the dysfunction of the endothelium as a common underlying causative factor. In this context, BH4 appears to be an alternative agent to the widely known scavenger molecules in reducing oxidative stress-induced vascular damage.


goto top of outline Acknowledgments

We thank Professor M. Taddei for the synthesis of the scrambled peptide.

This work was supported by funds from the Italian Ministry Association for Cancer Research (AIRC), the University of Siena (PAR 2000), the CAIN Foundation and the Gillson-Longenbaugh Foundation.

 goto top of outline References
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 goto top of outline Author Contacts

Prof. Marina Ziche
Pharmacology Section, Department of Molecular Biology
University of Siena
Via A. Moro 2, IT–53100 Siena (Italy)
Tel. +39 0577 234 444, Fax +39 0577 234 343, E-Mail ziche@unisi.it

 goto top of outline Article Information

Received: December 8, 2003
Accepted: December 22, 2003
Published online: March 19, 2004
Number of Print Pages : 6
Number of Figures : 2, Number of Tables : 1, Number of References : 15

 goto top of outline Publication Details

Journal of Vascular Research (Incorporating International Journal of Microcirculation)
Founded 1964 as Angiologica by M. Comèl and L. Laszt (1964–1973) continued as Blood Vessels by J.A. Bevan (1974–1991)
Official Journal of the European Society for Microcirculation

Vol. 41, No. 2, Year 2004 (Cover Date: March-April 2004)

Journal Editor: U. Pohl, Munich
ISSN: 1018–1172 (print), 1423–0135 (Online)

For additional information: http://www.karger.com/jvr

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